Facile and rapid access to linear and truncated microcystin analogues for the implementation of immunoassays

Abstract

A series of simplified microcystin-LR analogues based on Adda [(2S,3S,8S,9S,4E,6E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldecadienoic acid] or its corresponding aldol precursor linked to a polypeptide moiety have been synthesised and assessed for their binding affinity by the monoclonal antibody mAb MC159, an anti-microcystin-LR mAb recently selected by us for the detection of microcystins through various immunoassay formats. Some modifications have been brought to the enantiospecific synthesis of N-Boc-Adda developed by Pearson et al. (Org. Lett., 2000, 2, 2901) which enabled us to access in an economical and time-saving manner a small library of MC-LR linear analogues. Among which Adda was chosen to synthesise, as an illustrative example, a fluorescent probe derived from this beta-amino acid. This probe was subsequently solid-phase immobilised by means of oxime ligation in order to lead to biochips suitable for microcystin detection through the SPIT-FRI method.

Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry

“…Several approaches for isotope incorporation are available, including synthetic, conjugative or by exchange reaction with the intact molecule. While the synthetic route involves straightforward coupling of 13 C- or 14 N-containing amino acids, a major difficulty lies in preparation of the ADDA amino acid which contains four chiral centers, representing a significant synthetic undertaking [48, 49]. Thus, chemical synthesis methodologies for MC standards are time and economically impractical since the measurement of endogenous MC congeners would require the separate synthesis of each variant.…”

Synthesis and reactivity of a bis-sultone cross-linker for peptideconjugation and [18F]-radiolabelling via unusual “double click” approach

“…N a -(tert-Butyloxycarbonyl)-L-lysine carboxamide (Boc-Lys-NH 2 ) was prepared from commercially available N a -( tert -butyloxycarbonyl ) -N e -( benzyloxycarbonyl ) -L -lysine (Boc-Lys(Z)-OH, Bachem or Fluka), by using a conventional two-step synthetic procedure: aminolysis of mixed anhydride formed in situ by short treatment with isobutyl chloroformate and N-methylmorpholine (NMM), followed by hydrogenolysis to remove the Z protecting group. 23 The synthesis of dodecapeptides (N a -Ac-lysine-or aminooxyacetic acid-terminated) was carried out on an Applied Biosystems 433A peptide synthesizer using standard Fmoc/tBu chemistry 24 and Wang resin (Iris Biotech, loading 0.9 mmol g -1 ) on a scale of 0.25 mmol. The HPLCgradient grade acetonitrile (CH 3 CN) and methanol (CH 3 OH) were obtained from VWR.…”

A Simple and Fast Non-Radioactive Bridging Immunoassay for Insulin Autoantibodies

“…A microcystin-LR analogue N -Boc-Adda [(2 S ,3 S ,8 S ,9 S ,4 E ,6 E )-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldecadienoic acid] named GC300 was synthesized as previously described [10]. Ester-active GC300 was conjugated to insulin via its NH 2 function.…”